An ADAM (a disintegrin and metalloprotease) is a transmembrane protein that contains both a disintegrin and a metalloprotease domain (Primakoff et al., 2000). Therefore, ADAMs have both cell adhesion and protease activities. Not surprisingly, a variety of physiological functions have been identified or implicated for ADAMs, including shedding, fertilization, signal transduction and Alzheimer's disease.
For example, ADAMs 17, 10 and 9 have been found to participate in shedding, the release of extracellular domains of membrane-anchored proteins after being cleaved from the remaining part of the proteins by proteases (called sheddases or secretases). One of the best-studied examples of shedding is the release of tumor necrosis factor α (TNF-α), a cytokine involved in the inflammatory response.
TNF-α is synthesized as a 26 kDa membrane-anchored protein, from which a soluble 17 kDa extracellular domain is proteolytically released as active TNF-α. This proteolytic release is catalyzed by TNF-α converting enzyme (TACE or ADAM 17), which also catalyzes the proteolytic cleavage of many other substrates. In fact, cells derived from ADAM 17 knockout mice are also unable to shed the TNF receptor, the adhesion molecule L-selectin, or the Alzheimer's disease amyloid protein, indicating that these proteins are the proteolytic substrates of ADAM 17 as well. The knockout mice themselves would die in utero due to failure to shed embryonic transforming growth factor α.
Similarly, other ADAMs have also been found or predicted to play important roles in normal physiological functions, since the proper proteolytic processing of many cytokines, growth factors, receptors, adhesion molecules and enzymes relies on ADAMs.
Recently, it has also been shown that ADAMs are involved in the signal transduction responses of certain G-protein coupled receptors (GPCRs). As shown in FIG. 1, upon ligand binding to a GPCR, ADAM 10 is activated to cleave the epidermal growth factor (EGF) precursor, PRO-EGF, to mature and active EGF. The resulting EGF, in turn, binds to the EGF receptor and leads to EGF receptor transactivation as well as the subsequent mitogenic signaling response (Daub et al., 1996; Prenzel et al., 1999; Yan et al., 2000). Given the fact that ADAMs participate in many fundamental cellular functions, it is important to be able to determine ADAM activities quickly and efficiently. However, to date, ADAM activities have generally been assayed using traditional biochemical methods, including purification or partially purification of ADAMs from cells, followed by mixing with a substrate and determination of the amount of product produced (see e.g. Rosendahl et al., 1997). These methods are time consuming, require large numbers of cells, and may introduce artifacts during the process of purification. Therefore, an improved method for assaying ADAM activities is desirable.